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Altered cytochrome c display precedes apoptotic cell death in Drosophila.

Varkey J, Chen P, Jemmerson R, Abrams JM - J. Cell Biol. (1999)

Bottom Line: We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis.In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive.Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

ABSTRACT
Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

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Mitochondria from rpr- or grim-expressing cells promote caspase activation in vitro. Expression of rpr or grim triggers activation of caspase activity in the cytosol (S-100 fraction).  Activity is assayed by cleavage of PARP and detection of a signature 85-kD fragment (lanes 2 and 3). PARP-cleavage activity is  not detected in cytosolic preparations from parental SL2 cells  (lane 1). After incubation with mitochondrial fractions from rpr-  or grim-expressing cells (see Materials and Methods) the otherwise silent cytosolic S-100 fraction from SL2 cells now shows pronounced PARP cleavage activity (lanes 5 and 6). In contrast,  mitochondria from nonapoptotic SL2 cells, tested in parallel,  failed to promote caspase activation of the same cytosolic fraction (lane 4). L, SL2 cells; R, rpr-expressing cells; G, grim-expressing cells.
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Figure 5: Mitochondria from rpr- or grim-expressing cells promote caspase activation in vitro. Expression of rpr or grim triggers activation of caspase activity in the cytosol (S-100 fraction). Activity is assayed by cleavage of PARP and detection of a signature 85-kD fragment (lanes 2 and 3). PARP-cleavage activity is not detected in cytosolic preparations from parental SL2 cells (lane 1). After incubation with mitochondrial fractions from rpr- or grim-expressing cells (see Materials and Methods) the otherwise silent cytosolic S-100 fraction from SL2 cells now shows pronounced PARP cleavage activity (lanes 5 and 6). In contrast, mitochondria from nonapoptotic SL2 cells, tested in parallel, failed to promote caspase activation of the same cytosolic fraction (lane 4). L, SL2 cells; R, rpr-expressing cells; G, grim-expressing cells.

Mentions: Studies described in the previous section established that, although clearly altered, Drosophila cytochrome c is retained in the mitochondrial compartment during apoptosis. Since cell-free studies in vertebrate systems implicate mitochondrial factors (cytochrome c and/or other proteins) in the activation of some caspase enzymes (Liu et al., 1996; Zamzami et al., 1996; Evans et al., 1997; Kluck et al., 1997b; Kroemer et al., 1997) we sought to determine whether mitochondria from our insect model might exhibit similar properties. To test this possibility, we measured caspase activation in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Fig. 5 illustrates detection of caspase activation, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay (Bose et al., 1998) is indistinguishable from the signature activity reported in many mammalian systems (Lazebnik et al., 1994; Nicholson and Thornberry, 1997; Villa et al., 1997) and is readily detected in the cytosol of pre-apoptotic cells (Fig. 5, lane 2 induced for rpr; lane 3 induced for grim) but not in cytosol from parental L2 (Fig. 5, lane 1). Lanes 5 and 6 of Fig. 5 show that mitochondria isolated from rpr- or grim-expressing cells trigger the appearance of PARP cleavage activity in the otherwise silent L2 cell cytosol. In contrast, mitochondria from parental L2 cells did not provoke similar cleavage of PARP (Fig. 5, lane 4). These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim.


Altered cytochrome c display precedes apoptotic cell death in Drosophila.

Varkey J, Chen P, Jemmerson R, Abrams JM - J. Cell Biol. (1999)

Mitochondria from rpr- or grim-expressing cells promote caspase activation in vitro. Expression of rpr or grim triggers activation of caspase activity in the cytosol (S-100 fraction).  Activity is assayed by cleavage of PARP and detection of a signature 85-kD fragment (lanes 2 and 3). PARP-cleavage activity is  not detected in cytosolic preparations from parental SL2 cells  (lane 1). After incubation with mitochondrial fractions from rpr-  or grim-expressing cells (see Materials and Methods) the otherwise silent cytosolic S-100 fraction from SL2 cells now shows pronounced PARP cleavage activity (lanes 5 and 6). In contrast,  mitochondria from nonapoptotic SL2 cells, tested in parallel,  failed to promote caspase activation of the same cytosolic fraction (lane 4). L, SL2 cells; R, rpr-expressing cells; G, grim-expressing cells.
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Related In: Results  -  Collection

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Figure 5: Mitochondria from rpr- or grim-expressing cells promote caspase activation in vitro. Expression of rpr or grim triggers activation of caspase activity in the cytosol (S-100 fraction). Activity is assayed by cleavage of PARP and detection of a signature 85-kD fragment (lanes 2 and 3). PARP-cleavage activity is not detected in cytosolic preparations from parental SL2 cells (lane 1). After incubation with mitochondrial fractions from rpr- or grim-expressing cells (see Materials and Methods) the otherwise silent cytosolic S-100 fraction from SL2 cells now shows pronounced PARP cleavage activity (lanes 5 and 6). In contrast, mitochondria from nonapoptotic SL2 cells, tested in parallel, failed to promote caspase activation of the same cytosolic fraction (lane 4). L, SL2 cells; R, rpr-expressing cells; G, grim-expressing cells.
Mentions: Studies described in the previous section established that, although clearly altered, Drosophila cytochrome c is retained in the mitochondrial compartment during apoptosis. Since cell-free studies in vertebrate systems implicate mitochondrial factors (cytochrome c and/or other proteins) in the activation of some caspase enzymes (Liu et al., 1996; Zamzami et al., 1996; Evans et al., 1997; Kluck et al., 1997b; Kroemer et al., 1997) we sought to determine whether mitochondria from our insect model might exhibit similar properties. To test this possibility, we measured caspase activation in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Fig. 5 illustrates detection of caspase activation, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay (Bose et al., 1998) is indistinguishable from the signature activity reported in many mammalian systems (Lazebnik et al., 1994; Nicholson and Thornberry, 1997; Villa et al., 1997) and is readily detected in the cytosol of pre-apoptotic cells (Fig. 5, lane 2 induced for rpr; lane 3 induced for grim) but not in cytosol from parental L2 (Fig. 5, lane 1). Lanes 5 and 6 of Fig. 5 show that mitochondria isolated from rpr- or grim-expressing cells trigger the appearance of PARP cleavage activity in the otherwise silent L2 cell cytosol. In contrast, mitochondria from parental L2 cells did not provoke similar cleavage of PARP (Fig. 5, lane 4). These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim.

Bottom Line: We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis.In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive.Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

ABSTRACT
Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

Show MeSH